The prevalence of mutations in the GJB2 gene encoding connexin26 (Cx26) in sensorineural deafness underscores the critical importance of this protein in cochlear function. Cxs, uniquely among ion channels, have dual functional roles, as intercellular, gap junction (GJ) channels, which mediate direct electrical and chemical communication between cells, and as undocked hemichannels (HCs), which mediate signaling across the plasma membrane. Despite a wealth of information obtained from studies of null mutations, the role of Cx26 in the cochlear sensory epithelium remains poorly understood. This is due, in part, to the fact that all functions for Cx26, including GJ- and HC-mediated, are abolished precluding assessment of GJ vs HC contributions as well as limiting investigation of mechanisms underlying cellular dysfunction. This proposal focuses on a growing number of functional variants of Cx26 that also cause sensorineural deafness, with hearing loss accompanied by neoplastic and infectious cutaneous manifestations resulting in their classification in one of several syndromes such as keratitis-ichthyosis-deafness (KID), a particularly severe affliction. KID mutants exhibit differential effects on Cx GJ channel and HC function and our studies are uncovering different aberrant functional properties that appear to underlie the notable phenotypic differences among patients. In this proposal, we build on our initial characterizations, investigating additional aberrant characteristics to gain insights into mechanisms of disease pathogenesis, genotype-phenotype associations as well as channel structure-function relationships pertaining to Cx channel permeation, gating and regulation. The common association of KID mutants with the aqueous pore suggests altered permeability characteristics are principal contributors to disease and we examine effects on permeability to Ca2+ and ATP, known key signaling molecules in the cochlea. Purinergic signaling, in particular, plays a prominent role in cochlear function and we investigate how KID mutants affect HC-mediated ATP release and subsequent signaling, teasing out GJ- and HC mediated components. We examine ATP signaling, first in exogenous expression systems, which permits quantification due to defined Cx expression and simple coupling geometries, but extend studies to native cells using cochlear explant cultures. Using a Tet-On inducible expression system, we will introduce KID mutants into cochlear supporting cells and examine effects on cell function, purinergic signaling and possible impact on hair cell survival. Finally, we use our extensive knowledge of GJ and HC structure/function and propose to generate variants of Cx26 specifically lacking function as a HC (Cx26-HC) without compromising biophysical properties of the GJ channel and conversely lacking function as a GJ channel (Cx26-GJ). Given the absence of Cx-specific blockers, these reagents would provide a unique means of unequivocally assessing contributions of GJ channels and HCs to cochlear function. Overall, we view that this work will have a major impact in understanding Cx-mediated hearing loss as well as a growing list of disorders in which aberrant Cx channels underlie tissue dysfunction.